PROTECTIVE ROLE OF SPECIFIC PATHOGEN FREE MICROBIOTA IN BILE DUCT LIGATED AND CCL4 TREATED MICE

Hepatitis Monthly

Volume 17 - Number Suppl.1

Article Type: Original Article
Abstract: Background: In chronic liver disease the presence of gut-derived bacterial products and the resultant increase in inflammatory cytokines in the splanchnic and systemic circulation may contribute to the progression of fibrosis. However, the composition of the intestinal microbiota and the host-microbe interaction in the development of liver fibrosis remain largely unknown. We hypothesized that fibrosis is attenuated in a gnotobiotic model of limited intestinal colonization (altered Schaedler flora, ASF) compared to a more complex colonization with specific pathogen free flora (SPF). We aimed to investigate the development of fibrosis and portal hypertension in ASF and SPF mice.
Methods: Liver fibrosis was induced by common bile duct ligation (BDL) for 14 days or intraperitoneal (IP) injection of 20% (dilution in olive oil) carbon tetrachloride (CCL4) for 10 weeks in ASF or SPF male, C57BL/6 mice. Hemodynamic measurements were performed after 14 days in BDL or 10 weeks in CCL4 treated mice. Liver histology and collagen deposition were evaluated using Sirius red staining for determination of fibrosis degree. To assess bacterial translocation, mesenteric lymph nodes, spleen and liver were dissected aseptically and then cultured on Luria Bertani or blood agar for aerobic and anaerobic culture respectively.
Results: There were no differences in portal pressure between sham-operated (controls) ASF or SPF mice. After BDL or CCL4 treatment portal pressure (PP), portosystemic shunts (PSS) and collagen deposition within the liver showed a significant increase in both groups. However, the increase in portal pressure and degree of fibrosis was higher in ASF than SPF mice. Results are as follow: in BDL mice, PP: ASF-Sham 8.34±1.1 cm H2O vs. SPF-Sham 7.18±1.3 cm H2O, p=0.15 and ASF-BDL (11.8± 1.1 cm H2O vs. SPF-BDL 9.75± 1.2 cm H2O, p=0.018). PSS%: ASF-sham (0.29 ±0.06% vs. SPF-sham 0.38 ±0.11%) or ASF-BDL (2.91±1.9% vs SPF-BDL 2.42± 2% p= 0.662). Collagen deposition%: ASF-sham 0.1% vs. SPF-sham 0.3% or ASF-BDL 9.8% vs. SPF-BDL 5.2% p=0.014). In CCL4 treated mice, PP: ASF-Sham 8.46 ± 0.05 cm H2O vs SPF-Sham 7.35 ±1.7 cm H2O, p=0.2 or ASF– CCL4 (12.15 ±1.6 cm H2O vs. SPF-CCL4 10.43 ± 1.2 cm H2O, p=0.04). PSS: ASF-control (0.62 ± 0.2%) vs SPF-control (0.49 ± 0.2%) or ASF-CCL4 (3.13±1.4%, p=0.015 vs. SPF-CCL4, 2.07±0.8%, p=0.015). Collagen deposition%: ASF-Sham 1.1% vs. SPF-Sham 0.8% and ASF-CCL4 (7.6±5% vs. SPF-CCL4 mice 4.3±2%, p= 0.02). Bacterial translocation was significantly higher in ASF-BDL than SPF-BDL mice suggesting that bacterial translocation occurred more frequently in ASF-BDL mice. The increase in the bile infarcts area was significantly higher in ASF mice (ASF-BDL 13.5% vs. SPFBDL 4.8% P=0.026). No significant bacterial translocation was observed in CCL4 treated mice.
Conclusions: SPF mice presented attenuated fibrosis and portal hypertension compared to ASF mice. Contrary to our hypothesis, these findings suggest that a more complex intestinal bacterial flora may play a hepato-protective role.Our results are in line with studies showing that germ free mice are more susceptible to liver injury and fibrosis suggesting the beneficial role of intestinal microbiota in preventing liver injury.
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